Field validation and assessment of an enzyme-linked immunosorbent assay for detecting chronic wasting disease in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni).

Tissue samples (n = 25,050 total) from 23,256 mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and white-tailed deer (Odocoileus virginianus) collected statewide in Colorado were examined for chronic wasting disease (CWD) using an enzyme-linked immunosorbent assay developed by Bio-Rad Laboratories, Inc. (brELISA), in a 2-phase study. In the validation phase of this study, a total of 4,175 retropharyngeal lymph nodes (RLN) or obex (OB) tissue samples were examined independently by brELISA and immunohistochemistry (IHC). There were 137 IHC-positive samples and 4,038 IHC-negative samples. Optical density (OD) values from brELISA were classified as "not detected" or "suspect" based on recommended cutoff values during the validation phase. Using IHC-positive cases as known CWD-infected individuals and assuming IHC-negative cases as uninfected, the relative sensitivity of brELISA depending on species ranged from 98.3% to 100% for RLN samples and 92.1% to 93.3% for OB samples; the relative specificity of brELISA depending on species ranged from 99.9% to 100% for RLN samples and was 100% for OB samples. Overall agreement between brELISA and IHC was > or = 97.6% in RLN samples and > or = 95.7% in OB samples of all species where values could be calculated; moreover, mean brELISA OD values were > or = 46X higher in IHC-positive samples than in IHC-negative samples. Discrepancies were observed only in early-stage cases of CWD. Based on the validation phase data, only RLN samples were collected for the field application phase of this study and only samples with brELISA OD values > 0.1 were examined by IHC. Among 20,875 RLN samples screened with brELISA during this second testing phase, 155 of 8,877 mule deer, 33 of 11,731 elk, and 9 of 267 white-tailed deer samples (197 total) had OD values > 0.1 and were further evaluated by IHC to confirm evidence of CWD infection. Of cases flagged for IHC follow-up, 143 of 155 mule deer, 29 of 33 elk, and all 9 white-tailed deer were confirmed positive. Mean (+/- SE) OD values for IHC-positive cases detected during the field application phase were comparable with those measured in RLN tissues during the validation phase. Based on these data, brELISA was determined to be an excellent rapid test for screening large numbers of samples in surveys designed to detect CWD infections in deer and elk populations.     

Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: final report

Final observations on experimental transmission of chronic wasting disease (CWD) from elk (Cervus elaphus nelsoni) and white-tailed deer (Odocoileus virginianus) to fallow deer (Dama dama) are reported herein. During the 5-year study, 13 fawns were inoculated intracerebrally with CWD-infected brain material from white-tailed deer (n = 7; Group A) or elk (n = 6; Group B), and 3 other fawns were kept as uninoculated controls (Group C). As described previously, 3 CWD-inoculated deer were euthanized at 7.6 mo post-inoculation (MPI). None revealed presence of abnormal prion protein (PrP(d)) in their tissues. At 24 (Group A) and 26 (Group B) MPI, 2 deer were necropsied. Both animals had a small focal accumulation of PrP(d) in their midbrains. Between 29 and 37 MPI, 3 other deer (all from Group A) were euthanized. The 5 remaining deer became sick and were euthanized between 51 and 60 MPI (1 from Group A and 4 from Group B). Microscopic lesions of spongiform encephalopathy (SE) were observed in only these 5 animals; however, PrP(d) was detected in tissues of the central nervous system by immunohistochemistry, Western blot, and by commercial rapid test in all animals that survived beyond 24 MPI. This study demonstrates that intracerebrally inoculated fallow deer not only amplify CWD prions, but also develop lesions of spongiform encephalopathy.     

Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route.

This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years after inoculation. During that time, abnormal prion protein (PrP(res)) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry and Western blot. However, microscopic lesions suggestive of spongiform encephalopathy (SE) in the brains of these PrP(res)-positive animals were subtle in 3 cases and absent in 2 cases. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46, and S at codon 146 in all samples. Findings of this study show that although PrP(res) amplification occurred after direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of SE. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrP(res). Although intracerebral inoculation is an unnatural route of exposure, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum but also may not result in amplification of PrP(res) within CNS tissues during the normal lifespan of cattle.     

Comparison of two automated immunohistochemical procedures for the diagnosis of scrapie in domestic sheep and chronic wasting disease in North American white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus).

Two commercially available automated immunohistochemistry platforms, Ventana NexES and DakoCytomation Autostainer Universal Staining System, were compared for diagnosing sheep scrapie and cervid chronic wasting disease. Both automated platforms used the same antiprion protein monoclonal primary antibodies, but different platform-specific linker and amplification reagents and procedures. Duplicate sections of brainstem (at the level of the obex) and lymphoid tissue (retropharyngeal lymph node or tonsil) from the same tissue block were immunostained for the comparison. Examination of 1,020 tissues from 796 sheep revealed 100% concordance of results between the Ventana NexES and DakoCytomation platforms for diagnosing sheep scrapie from lymphoid tissue (103/103 positive; 405/405 negative) and brainstem (120/120 positive; 392/392 negative). Similarly, examination of 1,008 tissues from 504 white-tailed deer revealed 100% concordance between the Ventana NexES and DakoCytomation platforms for diagnosing chronic wasting disease from lymphoid tissue (104/104 positive; 400/400 negative) and brainstem (104/104 positive; 400/400 negative). Examination of 1,152 tissues from 482 mule deer revealed a concordance of 98.6% in lymphoid tissue and 99.9% in brainstem between the Ventana NexES and DakoCytomation platforms for diagnosing chronic wasting disease. The results indicate equivalence or near equivalence between the DakoCytomation and Ventana NexES autostainer platforms for identification of the disease-associated prion protein (PrPd)-positive and PrPd-negative brain and lymphoid tissues in sheep, white-tailed deer, and mule deer.     

Preliminary observations on the experimental transmission of scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation

To determine the transmissibility of scrapie to Rocky Mountain elk (Cervus elaphus nelsoni), six elk calves were inoculated intracerebrally with brain suspension from sheep naturally affected with scrapie. One elk developed a brain abscess and was euthanatized at 7 weeks postinoculation (PI), and two others died at 6 and 15 months PI because of physical injuries. At 25 and 35 months PI, two other elk died after brief terminal neurologic episodes. Necropsy of these revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of spongiform encephalopathy were seen throughout the brains and the spinal cords, and in both cases these tissues were positive for PrP(res) by immunohistochemistry. Brains of both animals were positive for PrP(res) by western blot and for scrapie-associated fibrils (SAFs) by negative stain electron microscopy. PrP(res) and SAFs were not detected in the three elk that died or were euthanatized because of coincidental causes. Over 3.5 years after initiation of this experiment, the one remaining inoculated elk and two uninoculated (control) elk are alive and apparently healthy. These preliminary findings demonstrate that 1) sheep scrapie agent can be transmitted to elk by intracerebral inoculation; 2) the infection can result in severe, widely distributed spongiform change and accumulations of PrP(res) in the central nervous system (CNS); and 3) based on the examination of a limited number of CNS sections from two cases, this condition cannot be distinguished from chronic wasting disease with currently available diagnostic techniques.     

Detection of PrP(CWD) in postmortem rectal lymphoid tissues in Rocky Mountain elk (Cervus elaphus nelsoni) infected with chronic wasting disease.

Preclinical diagnostic tests for transmissible spongiform encephalopathies have been described for mule deer (Odocoileus hemionus), using biopsy tissues of palatine tonsil, and for sheep, using lymphoid tissues from palatine tonsil, third eyelid, and rectal mucosa. The utility of examining the rectal mucosal lymphoid tissues to detect chronic wasting disease (CWD) was investigated in Rocky Mountain elk (Cervus elaphus nelsoni), a species for which there is not a live-animal diagnostic test. Postmortem rectal mucosal sections were examined from 308 elk from two privately owned herds that were depopulated. The results of the postmortem rectal mucosal sections were compared to immunohistochemical staining of the brainstem, retropharyngeal lymph nodes, and palatine tonsil. Seven elk were found positive using the brainstem (dorsal motor nucleus of the vagus nerve), retropharyngeal lymph nodes, and palatine tonsil. Six of these elk were also found positive using postmortem rectal mucosal sections. The remaining 301 elk in which CWD-associated abnormal isoform of the prion protein (PrP(CWD)) was not detected in the brainstem and cranial lymphoid tissues were also found to be free of PrP(CWD) when postmortem rectal mucosal sections were examined. The use of rectal mucosal lymphoid tissues may be suitable for a live-animal diagnostic test as part of an integrated management strategy to limit CWD in elk.     

Preliminary observations of genetic susceptibility of elk (Cervus elaphus nelsoni) to chronic wasting disease by experimental oral inoculation.

To compare the genetic susceptibility of elk (Cervus elaphus nelsoni) with various alleles of the PRNP gene, which encodes the normal cellular prion protein, to chronic wasting disease (CWD), eight 8-month-old elk calves of 3 genotypes (2 132MM, 2 132LM, and 4 132LL) were orally dosed with CWD-infected brain material from elk. During postinoculation (PI) month 23, both 132MM elk had lost appetite, developed clinical signs of weight loss and central nervous system (CNS) dysfunction, and were euthanized. Two other elk (both 132LM) developed similar clinical signs of disease and were euthanized during PI month 40. All 4 affected elk had microscopic lesions of spongiform encephalopathy (SE), and PrPres, the disease-associated form of the prion protein, was detected in their CNS and lymphoid tissues by use of immunohistochemical (IHC) and Western blot (WB) techniques. These findings indicate that elk with MM and LM at codon 132 are susceptible to orally inoculated CWD. All 4 LL elk are alive at PI year 4 and are clinically normal, which suggests that 132LL elk may have reduced susceptibility to oral infection with CWD-infected material or may have prolonged incubation time.     

Distribution of protease-resistant prion protein and spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease

Serial sections of brain and palatine tonsil were examined by immunohistochemical staining (IHC) using monoclonal antibody F89/160.1.5 for detecting protease-resistant prion protein (PrP(res)) in 35 hunter-killed mule deer (Odocoileus hemionus) with chronic wasting disease. Serial sections of brain were stained with hematoxylin and eosin and examined for spongiform encephalopathy (SE). Clinical signs of disease were not observed in any of these deer. On the basis of the location and abundance of IHC and the location and severity of SE, deer were placed into four categories. Category 1 (n = 8) was characterized by IHC in the palatine tonsil with no evidence of IHC or SE in the brain. Category 2 (n = 13) was characterized by IHC in the palatine tonsil and IHC with or without SE in the dorsal motor nucleus of the vagus nerve (DMNV). Category 3 (n = 2) was characterized by IHC in the palatine tonsil, IHC with SE in the myelencephalon, and IHC without SE in the hypothalamus. Category 4 (n = 12) was characterized by IHC in the palatine tonsil and IHC with SE throughout the brain. Category I may represent early lymphoid tissue localization of PrP(res). The DMNV appears to be the most consistent single neuroanatomic site of detectable PrP(res). Categories 2-4 may represent a progression of spread of PrP(res) and SE throughout the brain. IHC in tonsil and brain and SE in brain were not detected in 208 control deer.     

Variable patterns of distribution of PrP(CWD) in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease

Sections of medulla oblongata, taken at the level of the obex, palatine tonsil and medial retropharyngeal lymph node from 10,269 captive Rocky Mountain elk (Cervus elaphus nelsoni), were examined by immunohistochemical staining with monoclonal antibody for the prion protein associated with the transmissible spongiform encephalopathy of cervids, chronic wasting disease (PrP(CWD)). The protein was detected in 226 of them. On the basis of the anatomical location of the deposits in the brainstem of 183 elk, four distinct patterns of distribution of PrP(CWD) within the parasympathetic region of the dorsal motor nucleus of the vagus nerve and the adjacent nuclei were observed. Mild gross lesions of chronic wasting disease (serous atrophy of fat) were observed in only three elk, all with spongiform degeneration; the other elk were considered to be in the preclinical stage of the disease. In contrast with the relatively predictable distribution of prion protein (PrP) in the brain and cranial nodes of sheep and mule deer, the distribution of PrP(CWD) in the brain and nodes of the elk was more variable and unrelated to their PrP genotype. One hundred and fifty-five of the 226 positive elk had deposits of PrP(CWD) in the brainstem and lymphoid tissues, 43 had deposits only in the lymphoid tissue and 28 had deposits only in the brainstem.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in black-tailed deer (Odocoileus hemionus columbianus) and mule deer (Odocoileus hemionus hemionus)

Deer are considered important intermediate hosts for the coccidian parasites, Toxoplasma gondii and Neospora caninum. Antibodies to N. caninum and T. gondii were determined in sera of 42 mule deer (Odocoileus hemionus hemionus) and 43 black-tailed deer (Odocoileus hemionus columbianus) from Washington state, USA, using direct agglutination test with specific antigens. A titer of 1:25 was considered diagnostic for both parasites. N. caninum antibodies were found in 7 of 42 mule deer and 8 of 43 black-tailed deer. T. gondii antibodies were found in 14 black-tailed deer but not in any of the mule deer. This is probably the first report of seroprevalence of N. caninum in these hosts.     

Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): Early detection and late stage distribution of protease-resistant prion protein

Chronic wasting disease (CWD), an important emerging prion disease of cervids, is readily transmitted by intracerebral or oral inoculation from deer-to-deer and elk-to-elk, suggesting the latter is a natural route of exposure. Studies of host range susceptibility to oral infection, particularly of those species found in habitats where CWD currently exists are imperative. This report describes the experimental transmission of CWD to red deer following oral inoculation with infectious CWD material of elk origin. At 18 to 20 months post-inoculation, mild to moderate neurological signs and weight loss were observed and animals were euthanized and tested using 3 conventional immunological assays. The data indicate that red deer are susceptible to oral challenge and that tissues currently used for CWD diagnosis show strong abnormal prion (PrP^CWD) accumulation. Widespread peripheral PrP^CWD deposition involves lymphoreticular tissues, endocrine tissues, and cardiac muscle and suggests a potential source of prion infectivity, a means of horizontal transmission and carrier state.     

Abundant PrP(CWD) in tonsil from mule deer with preclinical chronic wasting disease.

A monoclonal antibody dot-blot assay was used to evaluate detergent lysates of tonsil tissue from mule deer to detect PrP(CWD), the marker for the cervid transmissible spongiform encephalopathy chronic wasting disease (CWD). Samples of formalin-fixed brain and tonsil tissues from mule deer were examined for PrP(CWD) using immunohistochemistry (IHC) with Mab F99/97.6.1, the gold standard for diagnosis of preclinical CWD. The contralateral tonsil from each of the 143 deer was prepared for confirmatory IHC and as a 10% (wt/vol) detergent lysate without purification or enrichment steps for monoclonal antibody dot-blot assay. PrP(CWD) was detected by dot-blot assay in 49 of 50 samples considered positive by IHC. Forty-eight of the positive samples were evaluated with a quantitative dot-blot assay calibrated with recombinant PrP. Tonsillar PrP(CWD) concentrations ranged from 34 to 1,188 ng per 0.5 mg starting wet weight of tissue. The abundant PrP(CWD) in mule deer tonsil will facilitate development and validation of high-throughput screening tests for CWD in large populations of free-ranging deer.     

Transmission of sheep scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation: final outcome of the experiment.

This is a final report of an experimental transmission of sheep scrapie agent by intracerebral inoculation to Rocky Mountain elk (Cervus elaphus nelsoni). It documents results obtained in experimental (n = 6) and control (n = 2) elk. During the first 2 years postinoculation (PI), 3 animals died or were euthanized because of infection or injuries other than spongiform encephalopathy (SE). In years 3 and 4 PI, 3 other inoculated elk died after brief terminal neurological episodes. Necropsy of these animals revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of SE were seen throughout the brain and spinal cord, and the tissue was positive for proteinase K-resistant prion protein (PrPres) by immunohistochemistry (IHC) and by Western blot. Scrapie-associated fibrils (SAF) were observed by negative-stain electron microscopy in the brain of elk with neurologic signs. PrPres and SAF were not detected in the 3 inoculated elk necropsied during the first 2 years or in the 2 control animals. Retrospective analysis of the gene-encoding cervid PrP revealed a polymorphism at codon 132. The elk with SE were either homozygous (MM) or heterozygous (LM). These findings confirm that intracerebral inoculation of sheep scrapie agent results in SE with accumulations of PrPres in the central nervous system of elk. Based on morphologic and IHC findings, the experimentally induced SE cannot be distinguished from chronic wasting disease of elk with currently available diagnostic techniques.     

Susceptibility of male white-tailed deer (Odocoileus virginianus) to Brucella ovis infection

Infection with Brucella ovis was established by conjunctival instillation in 8 male white-tailed deer (Odocoileus virginianus). Infection was transient in young bucks, but persisted in bucks that were mature when inoculated. The deer were euthanatized and necropsied at various intervals after inoculation. Brucella ovis was recovered from a mature buck at necropsy on postinoculation day 429. Four deer had gross lesions and histopathologic changes of the epididymides. A mature noninfected buck confined for 7 months with an infected buck acquired infection and developed epididymal lesions.     

Experimental transmission of malignant catarrhal fever to red deer (Cervus elaphus)

Abstract

Malignant catarrhal fever was transmitted from affected to recipient red deer (Cervus eluphus) using bloodor lymphoid suspension as inoculum. Incubation periods ranged from 11 to 26 days. The disease was also transmitted using lymphoid suspension stored overnight at 4°C or at ?70°C for 8 months.

The experimental disease was characterised by fever, depression, anorexia, diarrhoea and dysentry. The course of the disease was approximately 96 hours.

Major lesions consisted of acute mesenteric lymphadenitis and acute haemorrhagic typhlitis and colitis. Lesions in the caecum and colon started as multifocal mucosal haemorrhages and progressed rapidly to massive mucosal haemorrhage.     

Experimental transmission of malignant catarrhal fever to red deer (Cervus elaphus)

Malignant catarrhal fever was transmitted from affected to recipient red deer (Cervus elaphus) using blood or lymphoid suspension as inoculum. Incubation periods ranged from 11 to 26 days. The disease was also transmitted using lymphoid suspension stored overnight at 4 degrees C or at -70 degrees C for 8 months. The experimental disease was characterised by fever, depression, anorexia, diarrhoea and dysentry. The course of the disease was approximately 96 hours. Major lesions consisted of acute mesenteric lymphadenitis and acute haemorrhagic typhlitis and colitis. Lesions in the caecum and colon started as multifocal mucosal haemorrhages and progressed rapidly to massive mucosal haemorrhage.     

Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus) with chronic wasting disease.

A new monoclonal antibody (MAb), F99/97.6.1, that has been used to demonstrate scrapie-associated prion protein PrP(Sc) in brain and lymphoid tissues of domestic sheep with scrapie was used in an immunohistochemistry assay for diagnosis of chronic wasting disease (CWD) in mule deer (Odocoileus hemionus). The MAb F99/97.6.1 immunohistochemistry assay was evaluated in brain and tonsil tissue from 100 mule deer that had spongiform encephalopathy compatible with CWD and from 1,050 mule deer outside the CWD-endemic area. This MAb demonstrated abnormal protease-resistant prion protein (PrP(res)) in brains of all of the 100 mule deer and in 99 of the 100 tonsil samples. No immunostaining was seen in samples collected from deer outside the endemic area. MAb F99/97.6.1 demonstrated excellent properties for detection of PrP(res) in fresh, frozen, or mildly to moderately autolytic samples of brain and tonsil. This immunohistochemistry assay is a sensitive, specific, readily standardized diagnostic test for CWD in deer.     

Comparison of retropharyngeal lymph node and obex region of the brainstem in detection of chronic wasting disease in white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) in Wisconsin was first identified in February 2002. By April 2005, medial retropharyngeal lymph node (RLN) tissues had been examined from over 75,000 white-tailed deer for the presence of CWD by either immunohistochemical (IHC) staining for the prion protein associated with CWD (PrP(res)) or by using enzyme-linked immunosorbent assays with confirmation of positives by IHC staining and had been detected in 469 animals. Obex tissue was also available from 438 of the CWD-positive animals and was CWD positive by IHC staining in 355 (81%). To verify whether false-negative results were possible examining only RLN, both obex and RLN samples were examined for CWD by IHC staining from 4,430 of the white-tailed deer harvested from an area in Wisconsin where the overall deer CWD prevalence was approximately 6.2%. Two hundred and fourteen of the 269 positive deer (79.6%) had deposits of PrP(res) in both obex and lymphoid tissues, 55 (20.4%) had deposits only in lymphoid tissue, and there were no deer that had deposits only in obex.     

Comparison of histological lesions and immunohistochemical staining of proteinase-resistant prion protein in a naturally occurring spongiform encephalopathy of free-ranging mule deer (Odocoileus hemionus) with those of chronic wasting disease of captive mule deer

In this investigation, the nature and distribution of histologic lesions and immunohistochemical staining (IHC) of a proteinase-resistant prion protein were compared in free-ranging mule deer (Odocoileus hemionus) dying of a naturally occurring spongiform encephalopathy (SE) and captive mule deer dying of chronic wasting disease (CWD). Sixteen free-ranging deer with SE, 12 free-ranging deer without SE, and 10 captive deer with CWD were examined at necropsy. Tissue sections were stained with hematoxylin and eosin, and duplicate sections were stained with a monoclonal antibody (F89/160.1.5). Histological lesions in the free-ranging deer with SE and captive deer with CWD were found throughout the brain and spinal cord but were especially prominent in the myelencephalon, diencephalon, and rhinencephalon. The lesions were characterized by spongiform degeneration of gray matter neuropil, intracytoplasmic vacuolation and degeneration of neurons, and astrocytosis. IHC was found throughout the brain and retina of deer with SE and CWD. Positive IHC was found in lymphoid tissue of deer with SE and CWD. Histologic lesions and IHC were not found in multiple sections of integument, digestive, respiratory, cardiovascular, endocrine, musculoskeletal, and urogenital systems of deer with SE or CWD. Comparison of histologic lesions and IHC in tissues of free-ranging deer with those of captive deer provides strong evidence that these two diseases are indistinguishable morphologically.